Project Summary Accumulating evidence demonstrates that the vascular endothelium plays an important role in the regulation of metabolic homeostasis. In accordance, endothelial dysfunction contributes to metabolic derangements associated with obesity and insulin resistance. Our laboratory provided the first evidence that endothelial cells (ECs) have the capacity to form lipid droplets (LDs) in vivo and demonstrated that LD turnover (i.e., synthesis and degradation) is necessary for maintaining EC quiescence and function. Specifically, pharmacological inhibition of key enzymes in the LD synthesis pathway, diacylglycerol acetyl transferase (DGAT) 1 and 2, led to endoplasmic reticulum (ER) stress. Alternatively, endothelial specific deletion of adipose triglyceride lipase (ATGL), the rate-limiting enzyme in LD hydrolysis, led to a profound reduction in endothelial nitric oxide synthase (eNOS) protein levels and nitric oxide (NO) bioavailability in standard chow fed mice. Indeed, these vascular perturbations have been linked to derangements in whole-body metabolism, but the role of EC LD turnover as it relates to the control of vascular and metabolic function is unknown. Therefore, in this proposal, I will define the specific role of endothelial LD turnover in maintaining vascular and metabolic function during normal physiology, DIO and in response to acute and chronic exercise. To achieve these ends, I will generate inducible, endothelial specific ATGL knockout (iECKO), DGAT1-iECKO and DGAT2-iECKO mice as tools to dissect the molecular mechanisms by which LD turnover controls vascular and whole-body metabolic homeostasis. In aim 1, I will dissect the cellular mechanisms by which disruption in LD turnover leads to endothelial dysfunction under normal physiology and DIO via rigorous and meticulous molecular and metabolic characterization. In aim 2, I will decipher the importance of endothelial LD turnover in maintaining insulin sensitivity in normal physiology and DIO via comprehensive in vivo metabolic studies and ex vivo tissue analysis. Lastly, in aim 3, I will establish the role of endothelial LD turnover in the regulation of adaptations in response to an acute bout of exercise bout and chronic endurance training using a similar approach as aim 2. Collectively, these studies will address a heretofore unknown role of EC LD turnover in whole-body metabolism. Results from these studies will provide a better understanding of the interrelationship between vascular and metabolic function during normal physiology and in the setting of obesity and type II diabetes. In addition to these research aims, this proposal describes a five-year intensive mentored training program with the goal of developing the Principal Investigator (PI, Nabil Boutagy, Ph.D.) into an independent and high impact, scientist in the fields of vascular biology and metabolism under the supervision of his primary mentor, Dr. William Sessa, and co-mentor, Dr. Gerald Shulman. Furthermore, a team of world-class scientists has been assembled to provide oversight and guidance in achieving all aspects of the proposed research and the PI?s career development.